DESCRIPTION: Expression of the recently discovered human restriction factor SAMHD1 is responsible for the infection block imposed to lentiviruses such as HIV-1, HIV-2 and SIVmac by primary macrophages, dendritic cells and resting CD4+ T-cells. SAMHD1 blocks lentiviral infection by preventing the occurrence of reverse transcription. The viral accessory protein Vpx, contained in SIVmac and HIV-2 particles, overcomes the SAMHD1 reverse transcription block by inducing SAMHD1 degradation. SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase that decreases the cellular levels of triphosphodeoxynucleotides (dNTPs). The dramatic decrease in dNTP levels in macrophages, dendritic cells and CD4+ resting T cells correlates with the inability of lentiviruses to undergo reverse transcription; therefore, SAMHD1 prevents lentiviral reverse transcription by depletion of dNTP levels. Interestingly, cycling and non-cycling cells express SAMHD1; however, SAMHD1's antiviral activity is only observed in non-cycling cells. Our preliminary findings correlate the lentiviral restriction phenotype observed in non-cycling cells with the phosphorylation state of SAMHD1.These results strongly suggested that phosphorylation regulates the antiviral activity of SAMHD1; therefore, this proposal will test the hypothesis that phosphorylation of SAMHD1 induces a conformational change that closes the active site of SAMHD1 domain resulting in an enzymatically and antivirally inactive SAMHD1 protein. The following specific aims will be used to address this hypothesis. Aim1 will explore the role of SAMHD1 phosphorylation in retroviral restriction. For this purpose, we will study restriction of SAMHD1 proteins where the phosphorylatable residues are replaced by either a phosphomimetic or non-phosphorylatable residue. This aim will also explore the nature of the kinase involved in the phosphorylation of SAMHD1. Aim 2 will explore the ability of Vpx to modulate SAMHD1 antiviral and enzymatic activities before SAMHD1 degradation. Aim 3 will explore the regulation of the antiviral and enzymatic activity of SAMHD1. Specifically, this aim will test the notion that SAMHD1 is regulated by a ball-and-chain mechanism. Overall, this proposal will establish phosphorylation as a new framework for understanding the antiviral properties of SAMHD1. Understanding the regulation of SAMHD1 is instrumental for the development of novel anti-HIV-1 vaccine strategies since overcoming SAMHD1 increases the adaptive immune response during infection of dendritic cells and macrophages. In addition, macrophages represent one of the most resilient HIV-1 reservoirs, so understanding the regulation of SAMHD1 antiviral properties could provide novel insides for the elimination of HIV-1 reservoirs.